lab 3

• What is the primary use of Hematoxylin and Eosin (H&E) staining?

  Diagnosis of diseases like cancer
• What information does H&E staining provide about tissue samples?

  Distribution, shape, and structure of cells
• What is the principle behind H&E staining?

  Chemical attraction between tissue and dye
• What are the two histological stains combined in H&E?

  Hematoxylin and eosin
• What color does hematoxylin stain the nucleus?

  Deep blue-purple
• What does eosin stain in tissue samples?

  Cytoplasm and extracellular matrix
• How does hematoxylin function as a dye?

  It binds with acidic components of tissue
• What is the role of eosin in H&E staining?

  Colors basic parts of tissue
• Why is xylene used in the staining process?

  To remove paraffin from slides
• What is the first step in the H&E staining protocol?

  Xylene for 10 minutes
• What is the purpose of using 1% acid alcohol in the staining process?

  Improves H&E stain quality
• What does the 0.2% ammonia do in the staining process?

  Acts as a bluing agent
• How long should the tissue be dipped in eosin?

  2 seconds
• What is the final step in the H&E staining protocol?

  Observe under the microscope
• What are the steps involved in the H&E staining procedure?

  1. Xylene for 10 minutes
  2. 100% ethanol for 2 minutes
  3. 100% methanol for 2 minutes
  4. 70% methanol for 2 minutes
  5. 50% methanol for 2 minutes
  6. 30% methanol for 2 minutes
  7. Rinse in sterile H2O for 5 minutes
  8. Hematoxylin for 10 minutes
  9. Rinse under running tap water
  10. Three dips in 1% acid alcohol
  11. Rinse in running tap water
  12. Ten seconds in 0.2% ammonia
  13. Rinse in running tap water
  14. Fifteen dips in 90% ethanol
  15. Eosin for 2 seconds
  16. Shake in 90% ethanol
  17. Repeat in another jar with 90% ethanol
  18. Shake in 100% ethanol
  19. Xylene for 3 minutes
  20. Apply DPX mounting medium and cover slip
  21. Observe under the microscope
• Why must the dehydration process be done gradually?

  To prevent tissue damage and distortion, so that cells are not destroyed
• What are the factors that affect staining?

  Thickness of tissue, temperature, and time left in stain
• What are the differences between using paraffin-embedded tissues and frozen tissues in H&E staining?

  • Paraffin-embedded:
  • Dehydrated and embedded in paraffin
  • Cut with a microtome
  • Requires xylene for clearing
  • Frozen tissues:
  • Cut on a cryostat
  • Fixed in alcohol
  • No need for xylene
• What can cause uneven H and E staining?

  contaminated staining solution, paraffin was is not properly removed by xylene